Crosslinking of base-modified RNAs by synthetic DYW-KP base editors implicates an enzymatic lysine as the nitrogen donor for U-to-C RNA editing

Abstract

Sequence-specific cytidine to uridine (C-to-U) and adenosine to inosine editing tools can alter RNA and DNA sequences and utilize a hydrolytic deamination mechanism requiring an active site zinc ion and a glutamate residue. In plant organelles, DYW-PG domain containing enzymes catalyze C-to-U edits through the canonical deamination mechanism. Proteins developed from consensus sequences of the related DYW-KP domain family catalyze what initially appeared to be uridine to cytidine (U-to-C) edits leading to this investigation into the U-to-C editing mechanism. The synthetic DYW-KP enzyme KP6 was found sufficient for C-to-U editing activity stimulated by the addition of carboxylic acids in vitro. Despite addition of putative amine/amide donors, U-to-C editing by KP6 could not be observed in vitro. C-to-U editing was found not to be concomitant with U-to-C editing, discounting a pyrimidine transaminase mechanism. RNAs containing base modifications were highly enriched in interphase fractions consistent with covalent crosslinks to KP6, KP2, and KP3 proteins. Mass spectrometry of purified KP2 and KP6 proteins revealed secondary peaks with mass shifts of 319 Da. A U-to-C crosslinking mechanism was projected to explain the link between crosslinking, RNA base changes, and the ∼319 Da mass. In this model, an enzymatic lysine attacks C4 of uridine to form a Schiff base RNA-protein conjugate. Sequenced RT-PCR products from the fern Ceratopteris richardii indicate U-to-C base edits do not preserve proteinaceous crosslinks in planta. Hydrolysis of a protonated Schiff base conjugate releasing cytidine is hypothesized to explain the completed pathway in plants.

Keywords: C-to-U RNA editing; Ceratopteris richardii; RNA crosslinking; U-to-C RNA editing; metalloenzymes.

Figure 1

At top, an image of Coomassie-stained SDS-PAGE gels loaded with 10 μg of recombinant KP6 (left) and KP2 (right) purification fractions. Each marker lane is labeled with the reference masses in kDa to compare to expected masses for KP6 (83.8 kDa) and KP2 (83.4 kD). Crude L, ClearL, IMAC, IMAC-C, IEX, and SEC represent crude lysate, cleared lysate, immobilized metal-affinity chromatography, concentrated immobilized metal affinity chromatography, ion-exchange chromatography, and size-exclusion chromatography fractions, respectively. At bottom, C-to-U and U-to-C RNA editing was assayed using appropriate substrates (listed at right) using the following protein concentrations: KP6 crude lysate (3.6 μg/μl); KP6 cleared lysate (3.72 μg/μl); KP6 IMAC (1.1 μg/μl); KP6 IEX (0.4 μg/μl); KP6 SEC (0.1 μg/μl); KP2 crude lysate (9.0 μg/μl); KP2 cleared lysate (7.7 μg/μl); KP2 IMAC (7.5 μg/μl); KP2 IEX (0.5 μg/μl); KP2 SEC (1.2 μg/μl). Sequence traces from individual reactions are shown.

Figure 2

Several carboxylic acids enhance C-to-U RNA editing catalyzed by recombinant KP6. Relative C-to-U RNA editing was assayed in reactions with 2 mM additions of 20 different carboxylic acids. Percent editing was calculated from three reactions per treatment. Calculated values were then related to percent editing calculated from a no treatment reaction in the same batch using the same protein fraction (% editing treatment/% editing no treatment ×100). Ion-exchange and size-exclusion chromatography purification fractions of KP6 were used for this analysis from the initial protein purification shown in Figure 1. An X-Y scatterplot displays relative editing values calculated for each reaction with error bars representing 1 standard deviation from the mean for the triplicate reactions. Reactions were performed at pH 7.7, and all molecules shown would predominantly be in the ionized form. C-to-U, cytidine to uridine; U-to-C, uridine to cytidine.

Figure 3

C-to-U RNA editing activity catalyzed by KP6 is not concomitant with U-to-C RNA editing. A, the sequences of the RNA substrates are shown to represent differences between substrates. RNA substrates with C and U nucleotides (in blue) at the RNA editing position (underlined) were constructed with both terminal AC5/CYC (red) and SK/KS (yellow) sequences. B, reactions containing combinations of two putative substrates at equimolar concentrations are represented by cartoon boxes. The green arrow highlights C-to-U RNA editing with the mean value and 1 standard deviation from the mean shown for triplicate reactions. A representative Sanger sequencing trace is displayed next to each substrate cartoon to show the extent of conversion with the editing site underlined. Reactions were performed using size-exclusion chromatography purification fractions in the presence of 2 mM of adipate. C-to-U, cytidine to uridine; U-to-C, uridine to cytidine.

Figure 4

KP6 activity attributed to U-to-C RNA editing is linked to RNA–protein crosslinking.A, the plasmid sequence is represented that once transcribed results in a U-target present in the 3′ UTR of the DYW-KP protein. B, a detailed flowchart shows three different experiments evaluating RNA editing from the same batch of three bacterial batch culture replicates A, B, and C. At top, the RNA purification from bacterial pellets proceeded using the techniques in the original report (20). Two different reverse transcriptase enzymes were compared (RevertAID RT versus Maxima RT). In the center, a diagram represents an experiment where RNA editing was calculated from RNAs isolated from the aqueous and interphase fractions of an organic phase separation. At bottom, a drawing represents isolation of RNAs from a denaturing immobilized metal affinity chromatography experiment in 6M guanidinium HCl. Mean percent RNA editing and one standard deviation from the mean were calculated from Sanger sequencing traces from three separate bacterial cultures. Representative images are displayed to the right of arrows with the editing site underlined. IMAC, immobilized metal affinity chromatography; IPTG, isopropyl β-d-1-thiogalactopyranoside; U-to-C, uridine to cytidine.

Figure 5

U-to-C editing in the model fern C. richardii is not linked to crosslinking. At left, a detailed flowchart describes an experiment used to examine RNA editing in RNAs from different fronds and present in the aqueous versus the interphase fractions of an organic phase separation. At right, representative Sanger sequencing electrophoretograms are shown with the editing site underlined above editing site labels over mean percent editing values calculated from three fronds from different plants (at top). One standard deviation from the mean is shown. Since RNAs from fronds A, B, and C were combined to increase yield, mean editing values are shown (at bottom right) from only two sequencing reactions using a forward and a reverse primer with the editing sites underlined. U-to-C, uridine to cytidine.

Figure 6

A proposed mechanism for U-to-C crosslinking consistent with the observation that base differences in the Sanger sequencing traces are concomitant with RNA–protein crosslinking. The mechanism design was influenced by presence of local zinc ions and a catalytic glutamate in the DYW domain active site and the requirement for lysine as the only amino acid capable of both RNA–protein crosslinking and observed altered base pairing. Though the reaction mechanism does not appear to complete in bacteria, the full mechanism projects how hydrolysis of a protonated Schiff base can release the edited cytidine. Images were constructed using ChemDraw 23.0.1. U-to-C, uridine to cytidine.